2.1. PEPTIDE LIBRARIES
The use of peptide libraries is well known in the art. Such peptide libraries have generally been constructed by one of two approaches. According to one approach, peptides have been chemically synthesized in vitro in several formats. For example, Fodor et al., 1991, Science 251: 767-773, describes use of complex instrumentation, photochemistry and computerized inventory control to synthesize a known array of short peptides on an individual microscopic slide. Houghten et al., 1991, Nature 354: 84-86, describes mixtures of free hexapeptides in which the first and second residues in each peptide were individually and specifically defined. Lam et al., 1991, Nature 354: 82-84, describes a "one bead, one peptide" approach in which a solid phase split synthesis scheme produced a library of peptides in which each bead in the collection had immobilized thereon a single, random sequence of amino acid residues. For the most part, the chemical synthetic systems have been directed to generation of arrays of short length peptides, generally fewer than about 10 amino acids or so, more particularly about 6-8 amino acids. Direct amino acid sequencing, alone or in combination with complex record keeping of the peptide synthesis schemes, is required to use these libraries.
According to a second approach using recombinant DNA techniques, peptides have been expressed in biological systems as either soluble fusion proteins or viral capsid fusion proteins.
A number of peptide libraries according to the second approach have used the M13 phage. M13 is a filamentous bacteriophage that has been a workhorse in molecular biology laboratories for the past 20 years. M13 viral particles consist of six different capsid proteins and one copy of the viral genome, as a single-stranded circular DNA molecule. Once the M13 DNA has been introduced into a host cell such as E. coli, it is converted into double-stranded, circular DNA. The viral DNA carries a second origin of replication that is used to generate the single-stranded DNA found in the viral particles. During viral morphogenesis, there is an ordered assembly of the single-stranded DNA and the viral proteins, and the viral particles are extruded from cells in a process much like secretion. The M13 virus is neither lysogenic nor lytic like other bacteriophage (e.g., .lambda.); cells, once infected, chronically release virus. This feature leads to high titers of virus in infected cultures, i.e., 10.sup.12 pfu/ml.
The genome of the M13 phage is .about.8000 nucleotides in length and has been completely sequenced. The viral capsid protein, protein III (pIII) is responsible for infection of bacteria. In E. coli, the pillin protein encoded by the F factor interacts with pIII protein and is responsible for phage uptake. Hence, all E. coli hosts for M13 virus are considered male because they carry the F factor. Several investigators have determined from mutational analysis that the 406 amino acid long pIII capsid protein has two domains. The C-terminus anchors the protein to the viral coat, while portions of the N-terminus of pIII are essential for interaction with the E. coli pillin protein (Crissman and Smith, 1984, Virology 132: 445-455). Although the N-terminus of the pIII protein has been shown to be necessary for viral infection, the extreme N-terminus of the mature protein does tolerate alterations. In 1985, George Smith published experiments reporting the use of the pIII protein of bacteriophage M13 as an experimental system for expressing a heterologous protein on the viral coat surface (Smith, 1985, Science 228: 1315-1317). It was later recognized, independently by two groups, that the M13 phage pIII gene display system could be a useful one for mapping antibody epitopes. De la Cruz et al., 1988, J. Biol. Chem. 263: 4318-4322 cloned and expressed segments of the cDNA encoding the Plasmodium falciparum surface coat protein into the pIII gene, and recombinant phage were tested for immunoreactivity with a polyclonal antibody. Parmley and Smith, 1988, Gene 73: 305-318 cloned and expressed segments of the E. coli .beta.-galactosidase gene in the pIII gene and identified recombinants carrying the epitope of an anti-.beta.-galactosidase monoclonal antibody. The latter authors also described a process termed "biopanning", in which mixtures of recombinant phage were incubated with biotinylated monoclonal antibodies, and phage-antibody complexes could be specifically recovered with streptavidin-coated plastic plates.
In 1989, Parmley and Smith, 1989, Adv. Exp. Med. Biol. 251:215-218 suggested that short, synthetic DNA segments cloned into the pIII gene might represent a library of epitopes. These authors reasoned that since linear epitopes were often .about.6 amino acids in length, it should be possible to use a random recombinant DNA library to express all possible hexapeptides to isolate epitopes that bind to antibodies.
Scott and Smith, 1990, Science 249:386-390 describe construction and expression of an "epitope library" of hexapeptides on the surface of M13. The library was made by inserting a 33 base pair Bgl I digested oligonucleotide sequence into an Sfi I digested phage fd-tet, i.e., fUSE5 RF. The 33 base pair fragment contains a random or "degenerate" coding sequence (NNK).sub.6 where N represents G, A, T or C and K represents G or T. The authors stated that the library consisted of 2.times.10.sup.8 recombinants expressing 4.times.10.sup.7 different hexapeptides; theoretically, this library expressed 69% of the 6.4.times.10.sup.7 possible peptides (20.sup.6). Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA 87: 6378-6382 also described a somewhat similar library of hexapeptides expressed as pIII gene fusions of M13 fd phage. PCT publication WO 91/19818 dated Dec. 26, 1991 by Dower and Cwirla describes a similar library of pentameric to octameric random amino acid sequences.
Devlin et al., 1990, Science, 249:404-406, describes a peptide library of about 15 residues generated using an (NNS) coding scheme for oligonucleotide synthesis in which S is G or C.
Christian and colleagues have described a phage display library, expressing decapeptides (Christian et al., 1992, J. Mol. Biol. 227:711-718). The starting DNA was generated by means of an oligonucleotide comprising the degenerate codons NN(G/T)!.sub.10 with a self-complementary 3' terminus. This sequence, in forming a hairpin, creates a self-priming replication site which could be used by T4 DNA polymerase to generate the complementary strand. The double-stranded DNA was cleaved at the Sfi I sites at the 5' terminus and hairpin for cloning into the fUSE5 vector described by Scott and Smith, supra.
Other investigators have used other viral capsid proteins for expression of non-viral DNA on the surface of phage particles. The protein pVIII is a major M13 viral capsid protein and interacts with the single stranded DNA of M13 viral particles at its C-terminus. It is 50 amino acids long and exists in approximately 2,700 copies per particle. The N-terminus of the protein is exposed and will tolerate insertions, although large inserts have been reported to disrupt the assembly of pVIII fusion proteins into viral particles (Cesareni, 1992, FEBS Lett. 307:66-70). To minimize the negative effect of pVIII fusion proteins, a phagemid system has been utilized. Bacterial cells carrying the phagemid are infected with helper phage and secrete viral particles that have a mixture of both wild-type and pVIII fusion capsid molecules. pVIII has also served as a site for expressing peptides on the surface of M13 viral particles. Four and six amino acid sequences corresponding to different segments of the Plasmodium falciparum major surface antigen have been cloned and expressed in the comparable gene of the filamentous bacteriophage fd (Greenwood et al., 1991, J. Mol. Biol. 220:821-827).
Lenstra, 1992, J. Immunol. Meth. 152:149-157 described construction of a library by a laborious process encompassing annealing oligonucleotides of about 17 or 23 degenerate bases with an 8 nucleotide long palindromic sequence at their 3' ends. This resulted in the expression of random hexa- or octa-peptides as fusion proteins with the .beta.-galactosidase protein in a bacterial expression vector. The DNA was then converted into a double-stranded form with Klenow DNA polymerase, blunt-end ligated into a vector, and then released as Hind III fragments. These fragments were then cloned into an expression vector at the C-terminus of a truncated .beta.-galactosidase to generate 10.sup.7 recombinants. Colonies were then lysed, blotted on nitrocellulose filters (10.sup.4 /filter) and screened for immunoreactivity with several different monoclonal antibodies. A number of clones were isolated by repeated rounds of screening and were sequenced.
Cull et al., 1992, Proc. Natl. Acad. Sci. USA 89:1865-1869 described a system in which random peptides were fused to the carboxy terminus of the lac repressor. The fusion proteins contained an intact lac amino terminus (which is responsible for specific binding of the lac repressor to the DNA sequences constituting the lac operator sites). The nucleotide sequences encoding the fusion protein were cloned into a plasmid containing copies of the lac operator site. Thus, when the fusion protein was expressed in bacteria, it became bound to the operator sites of the plasmid encoding it. This provided a physical linkage between the fusion protein and the gene encoding it. When bacteria containing the plasmid were screened with ligands for which it was desired to isolate binding partners, the fusion proteins comprising peptides that specifically bound to the ligand were isolated, carrying along the genes that encoded those fusion proteins.
A comprehensive review of various types of peptide libraries can be found in Gallop et al., 1994, J. Med. Chem. 37:1233-1251.
2.2. LIGANDS USED TO SCREEN PEPTIDE LIBRARIES
Screening of peptide libraries has generally been confined to the use of a restricted number of ligands. Most commonly, the ligand has been an antibody (Parmley and Smith, 1989, Adv. Exp. Med. Biol. 251:215-218;Scott and Smith, 1990, Science 249:386-390). In many cases, the aim of the screening is to identify peptides from the library that mimic the epitopes to which the antibodies are directed. Thus, given an available antibody, peptide libraries are excellent sources for identifying epitopes or epitope-like molecules of that antibody (Yayon et al., 1993, Proc. Natl. Acad. Sci. USA 90:10643-10647).
While previous studies have succeeded in identifying epitopes and epitope-like molecules from peptide libraries, it has not been realized in the prior art that this approach could be extended by using the identified epitopes in a further round of screening of a peptide library to identify antibody-like molecules.
When it has been desired to obtain antibody-like molecules, the prior art has employed peptide libraries that contain naturally occurring antibody sequences. This has probably been due to the fact that specific binding by antibodies is known to depend upon a complex structure involving various complementarity determining regions (CDRs), often from both heavy and light antibody chains. Short peptides would not be expected to mimic such structures and longer peptides were thought to be unsuitable for display in the most commonly used libraries.
McCafferty et al., 1990, Nature 348:552-554 used PCR to amplify immunoglobulin variable (V) region genes and cloned those genes into phage expression vectors. The authors suggested that phage libraries of V, diversity (D), and joining (J) regions could be screened with antigen. The phage that bound to antigen could then be mutated in the antigen-binding loops of the antibody genes and rescreened. The process could be repeated several times, ultimately giving rise to phage which bind the antigen strongly.
Marks et al., 1991, J. Mol. Biol. 222:581-597 also used PCR to amplify immunoglobulin variable (V) region genes and cloned those genes into phage expression vectors.
Kang et al., 1991, Proc. Natl. Acad. Sci. USA 88:4363-4366 created a phagemid vector that could be used to express the V and constant (C) regions of the heavy and light chains of an antibody specific for an antigen. The heavy and light chain V-C regions were engineered to combine in the periplasm to produce an antibody-like molecule with a functional antigen binding site. Infection of cells harboring this phagemid with helper phage resulted in the incorporation of the antibody-like molecule on the surface of phage that carried the phagemid DNA. This allowed for identification and enrichment of these phage by screening with the antigen. It was suggested that the enriched phage could be subject to mutation and further rounds of screening, leading to the isolation of antibody-like molecules that were capable of even stronger binding to the antigen.
Hoogenboom et al., 1991, Nucleic Acids Res. 19:4133-4137 suggested that naive antibody genes might be cloned into phage display libraries. This would be followed by random mutation of the cloned antibody genes to generate high affinity variants.
In the prior art, peptide libraries have been screened with receptors to identify receptor ligand-like peptides, but peptide libraries have not been considered useful for identifying such ligand-binding peptides as those that mimic receptors.
Bass et al., 1990, Proteins: Struct. Func. Genet. 8:309-314 fused human growth hormone (hGH) to the carboxy terminus of the gene III protein of phage fd. This fusion protein was built into a phagemid vector. When cells carrying the phagemid were infected with a helper phage, about 10% of the phage particles produced displayed the fusion protein on their surfaces. These phage particles were enriched by screening with hGH receptor-coated beads. It was suggested that this system could be used to develop mutants of hGH with altered receptor binding characteristics.
Lowman et al., 1991, Biochemistry 30:10832-10838 used an improved version of the system of Bass et al. described above to select for mutant hGH proteins with exceptionally high affinity for the hGH receptor. The authors randomly mutagenized the hGH-pIII fusion proteins at sites near the vicinity of 12 amino acids of hGH that had previously been identified as being important in receptor binding.
Balass et al., 1993, Proc. Natl. Acad. Sci. USA 90:10638-10642 used a phage display library to isolate linear peptides that mimicked a conformationally dependent epitope of the nicotinic acetylcholine receptor. This was done by screening the library with a monoclonal antibody specific for the conformationally dependent epitope. The monoclonal antibody used was thought to be specific to the acetylcholine receptor's binding site for its natural ligand, acetylcholine.
Citation or identification of any reference herein shall not be construed as an admission that such reference is available as prior art to the present invention.